Abstract: :
Purpose: Cellular response to oscillating glucose levels andglycated albumin in human ARPE–19 cells and in chickenembryo CAMs were investigated.Methods: ARPE–19 cells in culture and CAMs exposed toglycated albumin, anti–oxidants, and oscillating glucose.Digital microscopic images and densitometry of CAMs, westernblotting of hypoxia inducible factor 1–alpha (HIF) andsuperoxide dismutase (SOD–1), and ELISA of VEGF and nitrotyrosine(NT) were done.Results: Glycated albumin (250 and 500 mcg/ml) was found toincrease vessel density in the CAM assay and this effect wasattenuated by the addition of various anti–oxidants (d–alpha–tocopherol,N–acetylcysteine, MnTBAP). Short–term (8 hr) andlong–term (1 wk) exposure of ARPE–19 cells to glycatedalbumin increased expression of HIF–1alpha, a marker ofangiogenesis and altered glucose, as well as a target proteinfor HIF, the seminal angiogenesis factor VEGF. In addition toHIF and VEGF, long–term exposure to a lower dose of glycatedalbumin (100 mcg/ml) also increased levels of the oxidativestress marker NT in the ARPE–19 cells. Long–termexposure to glucose oscillating to mimic meals in a diabetic( a diabetic level of 10 mM glucose alternating with a post–prandiallevel of 15 mM glucose every 1.5 hr three times daily) alsoled to increased HIF and VEGF expression with only a small effecton SOD–1 and NT levels. Finally, long–term exposureto oscillating glucose and glycated albumin together resultedin a more pronounced increase in HIF, VEGF, and SOD–1expression with little difference in NT levels as compared toglycated albumin or oscillating glucose exposure alone.Conclusions:Glycated proteins and oscillating hyperglycemia,early metabolic changes associated with diabetes, caused anincrease in angiogenic signaling in ARPE–19 cells andblood vessel formation in the CAM assay, putatively throughthe generation of ROS. This suggests that HIF could be playingan earlier role in the pathogenesis of proliferative diabeticretinopathy than previously thought.